Disparate spinal and supraspinal opioid antinociceptive responses in beta-endorphin-deficient mutant mice.

The role of endogenous opioid systems in the analgesic response to exogenous opiates remains controversial. We previously reported that mice lacking the peptide neurotransmitter beta-endorphin, although unable to produce opioid-mediated stress-induced antinociception, nevertheless displayed intact antinociception after systemic administration of the exogenous opiate morphine. Morphine administered by a peripheral route can activate opioid receptors in both the spinal cord and brain. However, beta-endorphin neuronal projections are confined predominantly to supraspinal nociceptive nuclei. Therefore, we questioned whether the absence of beta-endorphin would differentially affect antinociceptive responses depending on the route of opiate administration. Time- and dose-response curves were obtained in beta-endorphin-deficient and matched wild-type C57BL/6 congenic control mice using the tail-immersion/withdrawal assay. Null mutant mice were found to be more sensitive to supraspinal (i.c.v.) injection of the micro-opioid receptor-selective agonists, morphine and D-Ala(2)-MePhe(4)-Gly-ol(5) enkephalin. In contrast, the mutant mice were less sensitive to spinal (i.t.) injection of these same drugs. Quantitative receptor autoradiography revealed no differences between genotypes in the density of mu, delta, or kappa opioid receptor binding sites in either the spinal cord or pain-relevant supraspinal areas. Thus we report that the absence of a putative endogenous ligand for the mu-opioid receptor results in opposite changes in morphine sensitivity between discrete areas of the nervous system, which are not simply caused by changes in opioid receptor expression.

Metabolic profiling of body fluids by proton NMR: self-poisoning episodes with paracetamol (acetaminophen).

1H NMR spectra of urine and plasma from subjects who had taken paracetamol (acetaminophen) at a therapeutic dose or in self-poisoning episodes (both fatal and nonfatal) are compared. They provide convenient metabolic profiles. For overdose cases, intense resonances corresponding to high levels of both drug and endogenous metabolites are observed. The ratios of glucuronide to sulfate conjugates are unusually high in urine from overdose cases. Elevated levels of the cysteinyl and N-acetyl cysteinyl conjugates reflect increased glutathione conjugation in the liver. The observed excretion of high levels of amino acids by overdose subjects is suggestive of drug-induced hepatic damage. No resonances for drug metabolites are detected in plasma samples. However, characteristic and abnormally intense resonances for the amino acids Phe, Tyr, His, Gln, Pro, Ala, Val, Lys, Met, Ser, and Thr are indicative of severe liver failure and disruption of normal deamination and transamination processes.

1H NMR studies of urine during fasting: excretion of ketone bodies and acetylcarnitine.

High-resolution 1H NMR spectroscopy has been applied to a study of urine from five normal human subjects during a 48-h period of fasting and for 22 h thereafter. The excretion rates of all three ketone bodies (acetoacetate, 3-D-hydroxybutyrate, and acetone), acetylcarnitine, creatinine, and sarcosine during this period were measured. Parallel increases in the excretion of the ketone bodies and acetylcarnitine were observed during fasting with little change in the output of creatinine and sarcosine.

1H, 13C NMR, and electronic absorption spectroscopic studies of the interaction of cyanide with aurothiomalate.

The reaction between cyanide and aurothiomalate (Autm) has been studied by 1H and 13C NMR spectroscopy and by uv spectroscopy. At cyanide:Autm ratios greater than or equal to 2, aurocyanide, [Au(CN)2]-, is the sole product but was also produced at lower ratios. Two intermediates were also identified. These were a mixed ligand complex, [tmAuCN]-, which accounted for over 80% of the gold at a ratio of cyanide to Autm of 1, and a bisthiomalato complex, [Autm2]-, which accounted for 6.8% of the total gold at this ratio of cyanide to Autm. The formation of these complexes may be significant in the antiarthritic activity of Autm since cyanide is produced by potential target cells such as polymorphonuclear leukocytes.

A high resolution proton nuclear magnetic resonance approach to the study of hepatocyte and drug metabolism. Application to acetaminophen.

1H spin echo NMR spectra of intact hepatocytes, isolated from rat liver, showed resonances for glucose, mobile fatty acids, and +N(CH3)3 groups including choline headgroups of phosphoglycerides. Spectra from extracts of the same cells contained many more well resolved resonances due to low Mr metabolites. These included signals for free amino acids, ketone bodies, glucose, lactate, and acetate. 1H NMR spectra from suspensions of intact hepatocytes incubated with acetaminophen showed no resonances for drug metabolites, although changes in sugar resonances were observed. However, spectra of extracts from acetaminophen-treated hepatocytes contained resonances for both acetaminophen itself and its major metabolites, the glucuronide and sulfate conjugates. Results on the extent of acetaminophen metabolism as measured by 1H NMR compared well with previously reported chromatographic studies. The rate of metabolism of acetaminophen by hepatocytes was much slower in 2H2O buffer compared to H2O buffer and selective deuteration of several metabolites including the ketone bodies, glucose, and acetaminophen glucuronide was observed. The deuteration of glucose C2H appeared to be due to futile cycling of the glycolytic pathway to at least fructose 6-phosphate, and incorporation of deuterium by the enzyme phosphoglucoisomerase. This work demonstrates that 1H NMR studies of intact hepatocytes and cell extracts together can provide considerable insight into the metabolism of acetaminophen in vitro. Little pretreatment of samples is required, results can be obtained rapidly, and both normal and drug metabolites can be observed simultaneously. Similar studies should be applicable to a wide range of other drugs.

Two-dimensional proton nuclear magnetic resonance "maps" of acetaminophen metabolites in human urine.

Two-dimensional proton-proton correlated NMR spectra of concentrated urine from a subject who had ingested a 1-g dose of acetaminophen are reported. These "maps" provide a considerable simplification of the spectrum in comparison with conventional one-dimensional NMR spectra. In the present case, peaks for all the major acetaminophen metabolites, including the L-cysteinyl conjugate, can be unambiguously assigned.

Urinary excretion of acetaminophen and its metabolites as studied by proton NMR spectroscopy.

Acetaminophen and its glucuronide, sulfate, N-acetyl-L-cysteinyl, and L-cysteinyl metabolites can be rapidly detected by 1H NMR spectroscopy of intact, untreated human urine. Study of the time course of excretion of these metabolites in five clinically normal men after ingestion of the usual 1-g therapeutic dose of the drug showed that the mean 24-h excretion of the drug and these metabolites as determined by NMR was 77.3% of the dose. Respective relative proportions of the above metabolites were 49.9%, 37.6%, 3.0%, and 9.5% (L-cysteinyl plus free drug). Excretion of some other metabolites in urine, including creatinine, citrate, hippurate, and sarcosine was measured concurrently. Excretion of creatinine and sarcosine was closely correlated.

Use of high-resolution proton nuclear magnetic resonance spectroscopy for rapid multi-component analysis of urine.

Numerous low-Mr metabolites--including creatinine, citrate, hippurate, glucose, ketone bodies, and various amino acids--have been identified in 400- and 500-MHz proton nuclear magnetic resonance (1H NMR) spectra of intact human urine. The presence of many of these was related to the specific condition of the donors: humans in different physiological states (resting, fasting, or post-exercise) and pathological conditions (e.g., diabetes mellitus, cadmium-induced renal dysfunction). We have also monitored the metabolism of simple nonendogenous compounds (methanol and ethanol) and of acetaminophen. The pH-dependencies of the NMR chemical shifts of some urine components are reported. Our studies show that high-resolution 1H NMR spectroscopy provides a fast, simple method for "fingerprint" identification of urinary compounds. In some cases, analytes can be quantified by standard additions or by comparing integrated peak areas for the metabolites with those for creatinine. Determinations of creatinine by 1H NMR spectroscopy compared well with those by an independent chemical assay based on the Jaffé reaction.

Hypoxic cell sensitization to radiation damage by a new radiosensitizer: cis-dichloro-bis(1-(2-hydroxyethyl)-2-methyl-5-nitroimidazole-N3)platinum(II) (flap).

A new, stable platinum coordination complex (FLAP) containing the 5-nitroimidazole, metronidazole, has been prepared and characterized. The square-planar platinum(II) complex has two metronidazole molecules and two chlorine atoms in the cis configuration. The properties of FLAP differ significantly from metronidazole alone or other platinum complexes tested in the same system. It has a low toxicity towards Chinese hamster ovary cells and is a very effective radiosensitizer toward hypoxic cells in vitro: a one-h pretreatment with a non-toxic dose of 50 microM gave an enhancement ratio of 2.4. No potentiation of aerated cells to X-irradiation damage was observed after a similar schedule of pretreatment at the higher dose of 100 microM FLAP.